Cyanoarylporphyrazine dyes: multimodal compounds for personalised photodynamic therapy.

Photodynamic therapy is known as an effective primary and adjuvant anticancer treatment. Compounds with improved properties or additional modalities are still needed to create an 'ideal' photosensitizer. In this article, we review cyanoarylporphyrazine dyes for photodynamic (anticancer) therapy that we have synthesised to date. The review provides information on the chemistry of cyanoarylporphyrazines, photophysical properties, cellular uptake features and the use of various carriers for selective delivery of cyanoarylporphyrazines to the tumour. The potential of cyanoarylporphyrazines as photodynamic anti-tumour agents also has been evaluated. The most interesting feature of cyanoarylporphyrazines is the dependence of the fluorescence quantum yield and excited state lifetime on the viscosity of the medium, which makes it possible to use them as viscosity sensors in photodynamic therapy. In the future, we expect that the unique combination of photosensitizer and viscosity sensor properties of cyanoarylporphyrazines will provide a tool for dosimetry and tailoring treatment regimens in photodynamic therapy to the individual characteristics of each patient.

Synergistic Effect of the Combined Action of Targeted and Photodynamic Therapy on HER2-Positive Breast Cancer.

Development of combined schemes for the treatment of oncological diseases is a promising strategy to improve the effectiveness of antitumor therapy. This paper shows the fundamental possibility of multiplying the antitumor effect by combining targeted and photodynamic therapy. It was demonstrated that sequential treatment of HER-2 positive breast cancer cells with the targeted toxin DARPin-LoPE and the photoactive compound photodithazine leads to a synergistic enhancement of their effect. In the future, this approach is intended to achieve the maximum therapeutic effect while minimizing the risks of negative side effects.

Photoluminescent Nanomaterials for Medical Biotechnology.

Creation of various photoluminescent nanomaterials has significantly expanded the arsenal of approaches used in modern biomedicine. Their unique photophysical properties can significantly improve the sensitivity and specificity of diagnostic methods, increase therapy effectiveness, and make a theranostic approach to treatment possible through the application of nanoparticle conjugates with functional macromolecules. The most widely used nanomaterials to date are semiconductor quantum dots; gold nanoclusters; carbon dots; nanodiamonds; semiconductor porous silicon; and up-conversion nanoparticles. This paper considers the promising groups of photoluminescent nanomaterials that can be used in medical biotechnology: in particular, for devising agents for optical diagnostic methods, sensorics, and various types of therapy.

Upconversion Nanoparticles Decorated with Polysialic Acid for Solid Tumors Visualization In Vivo.

Upconversion nanoparticles (UCNPs) are a promising nanoplatform for bioreagent formation for in vivo imaging, which emit UV and blue light under the action of near-infrared radiation, providing deep tissue penetration and maintaining a high signal-to-noise ratio. In the case of solid tumor visualization, the UCNP surface functionalization is required to ensure a long circulation time, biocompatibility, and non-toxicity. The effective UCNP accumulation in the solid tumors is determined by the disturbed architecture of the vascular network and lymphatic drainage. This work demonstrates an approach to the UCNP biofunctionalization with endogenous polysialic acid for in vivo bioreagent formation. Bioreagents possess a low level of nonspecific protein adsorption and macrophage uptake, which allow the prolongation of the circulation time in the bloodstream up to 3 h. This leads to an intense photoluminescent signal in the tumor.

Model of Ovarian Adenocarcinoma Spheroids for Assessing Photodynamic Cytotoxicity.

The aim of the study was to compare the relevance of ovarian adenocarcinoma spheroids with that of a monolayer culture for assessing photodynamic effect of the tetrakis(4-benzyloxyphenyl)tetracyanoporphyrazine photosensitizer. MATERIALS AND METHODS: The work was performed on SKOV-3 human ovary adenocarcinoma cells grown in vitro in a monolayer culture and in the form of tumor spheroids obtained using culture plates with ultra-low attachment. We determined the photoinduced toxicity of porphyrazine on a monolayer culture using the MTT assay; the effect on the spheroids was tested by assessing the dynamics of their growth. Cellular uptake of porphyrazine was analyzed by confocal microscopy. RESULTS: Porphyrazine has a pronounced photodynamic effect on SKOV-3 cells. When exposed to light at a dose of 20 J/cm2, the IC50 value 24 h after exposure was 2.3 µM for SKOV-3 monolayer culture. For the spheroids, the effect manifested after a latency period: significant growth retardation of the treated spheroids appeared no sooner than 5 and 9 days after exposure. Notably, no decrease in the initial size of the treated spheroids was observed under any of the photodynamic regimes. The penetration depth of porphyrazine into spheroids was 50-100 µm during 24 h incubation. CONCLUSION: The limited penetration of the photosensitizer into the body of spheroids and its predominant accumulation in the surface layers can be one of the key factors behind the significant differences in the photodynamic response between the surface and deep layers of a spheroid. For cells located close to the spheroid surface, the photodynamic effect is comparable to that for a monolayer culture, while in deeper layers, the cells remain viable and support/maintain the growth of the spheroid even under intense photo-exposure. The fact that the in vitro distribution is similar to the inhomogeneous accumulation of photosensitizers in tumors in vivo allows us to consider spheroids more relevant than a monolayer culture for studying photodynamic anti-tumor effects.

Hydrogen peroxide in the reactions of cancer cells to cisplatin.

BACKGROUND: Hydrogen peroxide (H2O2) is thought to be one of the key components involved in the responses of tumor cells to chemotherapy. The aim of this study was to reveal the pathways and the phases of cisplatin-induced cell death that are characterized by changes of H2O2 level. METHODS: The genetically encoded cytosolic fluorescent sensor HyPer2 was used for flow cytometric analysis of the cisplatin-induced changes in H2O2 level in HeLa Kyoto cells. Using a vital dye and the apoptotic markers PE Annexin V or TMRE the pathways and stages of cell death were investigated simultaneously with HyPer2 response. The H2O2 level was studied separately in viable and early apoptotic cells after 12, 18, 24 h's incubation with cisplatin at several concentrations with or without the scavenger of reactive oxygen species NAC. RESULTS: Cisplatin causes dose- and time-dependent increase of H2O2 level in TMRE-positive and PE Annexin V-negative cancer cells. The scavenging of ROS by NAC decreased the H2O2 level and restored cell viability. CONCLUSION: Н2О2 generation begins in cells that have already lost mitochondrial membrane potential but have not yet externalized phosphatidylserine. Prevention of apoptosis by NAC confirmed the role of H2O2 in apoptosis induction. GENERAL SIGNIFICANCE: This is the first time that the sensor HyPer2 has been used in parallel with apoptotic markers and vital dye to demonstrate the role of H2O2 in different stages and types of tumor cell death under chemotherapeutic action.

Nanoparticle-based drug delivery via RBC-hitchhiking for the inhibition of lung metastases growth.

Delivery of particle-based theranostic agents via their transportation on the surfaces of red blood cells, commonly referred to as RBC-hitchhiking, has historically been developed as a promising strategy for increasing the extremely poor blood circulation lifetime, primarily, of the large-sized sub-micron agents. Here, we show for the first time that RBC-hitchhiking can be extremely efficient for nanoparticle delivery and tumor treatment even in those cases when no circulation prolongation is observed. Specifically, we demonstrate that RBC-hitchhiking of certain small 100 nm particles, unlike that of the conventional sub-micron ones, can boost the delivery of non-targeted particles to lungs up to a record high value of 120-fold (and up to 40% of the injected dose). To achieve this remarkable result, we screened sub-200 nm nanoparticles of different sizes, polymer coatings and ζ-potentials and identified particles with the optimal RBC adsorption/desorption behavior. Furthermore, we demonstrated that such RBC-mediated rerouting of particles to lungs can be used to fight pulmonary metastases of aggressive melanoma B16-F1. Our findings could change the general paradigm of drug delivery for cancer treatment with RBC-hitchhiking. It is not the blood circulation lifetime that is the key factor for nanoparticle efficiency, but rather the complexation of nanoparticles with the RBC. The demonstrated technology could become a valuable tool for development of new strategies based on small nanoparticles for the treatment of aggressive and small-cell types of cancer as well as other lung diseases.

The Effect of the Targeted Recombinant Toxin DARPin-PE40 on the Dynamics of HER2-Positive Tumor Growth.

The development of targeted toxins based on non-immunoglobulin targeting molecules appears to be one of the most advanced approaches in the targeted therapy of malignant tumors with a high expression of the HER2 receptor. Earlier, we showed that the targeted toxin DARPin-PE40 consisting of the HER2-specific non-immunoglobulin polypeptide (the targeting module) and a fragment of Pseudomonas exotoxin A (the toxic module) exhibits an antitumor effect in vivo against the HER2-positive adenocarcinoma xenograft. In this work, an in-depth analysis of the effect of DARPin-PE40 on the growth dynamics of experimental xenograft tumors was carried out. DARPin-PE40 was shown to inhibit tumor growth at a dose of 25 and 50 µg/animal and to cause tumor node reduction at a dose of 80 µg/animal, followed by growth resumption at the end of therapy. An evaluation of the tumor growth dynamics revealed statistically significant differences in tumor volume in mice in the experimental groups compared to the control group. The results testify to the potential of using the created targeted toxin as an agent for the targeted therapy of HER2-overexpressing tumors.

Spheroids of HER2-Positive Breast Adenocarcinoma for Studying Anticancer Immunotoxins In Vitro.

Tumor response to therapeutic treatment is largely determined by its heterogeneity and the presence of intercellular junctions, hindering the penetration of large molecules deep into the three-dimensional structure of the tumor. In that context, 3D in vitro tumor models such as cancer cell spheroids are becoming increasingly popular. We obtained spheroids of human breast adenocarcinoma SKBR-3 overexpressing the HER2 cancer marker. The toxicity of HER2-targeted immunotoxin 4D5scFv-PE40 against spheroids was shown to be several orders of magnitude lower compared to a monolayer cell culture. The significant difference in the severity of the immunotoxin effect can be explained by the fact that it ineffectively penetrates the spheroid and predominantly influences the cells of the outer layers. The resulting tumor spheroid model can be used in development of drugs for targeted therapy as well as to study ways to improve the efficiency of anticancer agents by targeting cell-cell contacts.

Recombinant Immunotoxin 4D5scFv-PE40 for Targeted Therapy of HER2-Positive Tumors.

Recombinant immunotoxins are extremely promising agents for the targeted therapy of tumors with a certain molecular profile. In this work, we studied the properties of a new recombinant HER2-specific immunotoxin composed of the scFv antibody and a fragment of Pseudomonas exotoxin A (4D5scFv-PE40). High affinity of the immunotoxin for the HER2 tumor marker, its selective cytotoxicity against HER2-overexpressing cells, and its storage stability were demonstrated. The 50% inhibitory concentration (IC50) of the 4D5scFv-PE40 immunotoxin for HER2-overexpressing cancer cells was 2.5-3 orders of magnitude lower compared to that for CHO cells not expressing this tumor marker and was 2.5-3 orders of magnitude lower than IC50 of free PE40 for HER2-overexpressing cancer cells. These findings provide a basis for expecting in the long run high therapeutic index values of the 4D5scFv-PE40 immunotoxin for its use in vivo.

Far-red fluorescent cell line for preclinical study of HER2-targeted agents.

We have created novel HER2-overexpressing human ovarian adenocarcinoma cell line stably transfected with far-red fluorescent protein TurboFP635. Growth characteristics, adhesion capacity and morphology of the cells do not differ from that of parental cell line. The obtained cell line (SKOV-kat) was confirmed to have high level of expression of the tumor marker HER2 comparable to that of initial cell line SKOV-3. The SKOV-kat cell line is designed for further establishment of experimental fluorescent models of HER2-overexpressing human ovarian cancer that make it possible to optimize and reduce the cost of preclinical study of agents for treatment of this disease.

[Targeted Delivery of Quantum Dots to HER2-Expressing Tumor Using Recombinant Antibodies].

Targeted delivery of semiconductor quantum dots (Q Ds) to tumors overexpressing HER2 cancer marker has been. demonstrated on immunocompromised mice bearing human breast cancer xenografts. To obtain targeted QDs complexes we applied the approach based on the use of protein adaptor system, RNAase barnase and its inhibitor barstar. Specific binding to target cancer marker was achieved through bivalent fusion protein containing two fragments of4D5scFv recombinant antibody and a fragment of barnase. QDs were conjugated to barstar, and final assembly of targeted complexes was obtained through non-covalent specific interaction of barstar, attached to QD, and barnase, that is part of the recombinant targeting protein. The efficient delivery of QDs to HER2-expressing tumor demonstrates the possibilities and prospects of the approach for targeted delivery of nanoparticles to cancer cells in vivo as the way to improve the efficiency of diagnosis and promote development of therapies based on the use of nanoparticles.

Novel recombinant anti-HER2/neu immunotoxin: design and antitumor efficiency.

The novel HER2/neu-specific recombinant immunotoxin 4D5scFv-PE40 consisting of 4D5scFv antibody (targeting module) and Pseudomonas exotoxin A fragment (effector module) combined in a single polypeptide chain via a flexible linker has been expressed and purified. This immunotoxin conserves specificity and affinity that are characteristics of the parental antibody 4D5scFv and exhibits selective and strong cytotoxic effect against cancer cells overexpressing HER2/neu receptor. The results of the experiments both in vitro (in cell cultures) and in vivo (in tumor-bearing animals) demonstrate high potential of 4D5scFv-PE40 for targeted therapy of tumors overexpressing HER2/neu.

[Development of a new photosensitizer on the basis of ytterbium porphyrazine complex].

The tetraphenyltetracyanoporphyrazine complex of ytterbium has been studied as a potential photosensitizer for fluorescence diagnostics and photodynamic therapy (PDT) of cancer. It has been shown that the new compound has an intensive absorption and fluorescence in the "tissue optical window". In particular, the absorption maximum of the complex is at the wavelength of 590 nm, and the fluorescence emission maximum is at 640 nm. A strong fluorescence enhancement with a 50-fold increase in the quantum yield has been revealed in blood serum. The experiments on human cancer cells line have demonstrated that the complex penetrates the cells in vitro and is located around the nuclei. The biodistribution and pharmacokinetics of the complex in animals have been investigated in vivo by a new method of transillumination fluorescence imaging using a peculiar setup. It has been found that the period of maximum uptake of the complex in mouse cervical carcinoma is from 3 to 6 h after i.v. injection, with the half-life in the tumor being 24 h. However, the selectivity of the complex in the tumor is not high enough. The time of clearance from the body is about 48 h. The area of the strongest fluorescence in the abdominal cavity in in vivo images is anatomically recognized as the intestine. This indicates that the new compounds undergo mainly the hepatic clearance mainly. The conventional methods ex vivo (confocal microscopy and point spectroscopic measurements) have detected the largest content of the complex in the intestine, liver, skin and tumor tissue. In general, the optical characteristics of the ytterbium porphyrazine complex as well as the features of its interaction with biological objects make it promising drug candidate for the photodynamic therapy and/or fluorescence diagnostics of cancer. However, a search for other novel formulations possessing a higher tumor selectivity remains an urgent problem.

[An extracellular pH changes registration by confocal microscopy in higher plant at the excitation potentials generation].

Confocal microscopy technique was applied for registration of apoplast pH changes in the Cucurbita pepo seedling stem during generation of action potential and variation potential. Fluorescent dye FITC-dextran was used for pH-changes registration. Analysis of fluorescence images and fluorescence spectra showed, that FITC-dextran was localized in cell walls. Propagation of action potential and variation potential was accompanied by transient increase of fluorescence intensity, which indicated alkalization of cell walls. This transient alkalization is proposed to be caused by a temporary inactivation of the H(+)-pump in the plasma membrane.

[Use of optical coherence tomography for prognosis of the severity of oral mucositis].

The results of oral mucosa monitoring by optical coherence tomography (OCT) in the course of radiochemotherapy of 18 cases of oropharyngeal cancer are discussed. Damage to the mucosa was mainly assessed using contrast characteristics deterioration and reduction of epithelial layer thickness. Significant variation in OCT image characteristics was identified vs. mucositis grade and prognostic criteria for individual mucosal radiosensitivity worked out intact contrast on day 1 of mucositis should be interpreted as a sign of relatively mild complication. Blurred contrast would indicate mucositis stage III-IV development. Numerical analysis of OCT image patterns confirmed the contribution of the endothelial blood vessels and connecting tissue to radiation-induced damage to the mucosa.

[Differences in the functional activity of human neutrophilic granulocytes in their interactions with semiconductor quantum dots].

The uptake of quantum dots (QD-5 nm particles of CdSe/ZnS-mercaptoacetic acid) by human neutrophilic granulocytes was studied using the methods of scanning laser and scanning probe microscopy. The results show that the neutrophilic granulocytes may be subdivided into three subpopulations: (1) the cells with no uptake of QD (10.0 +/- 2.0%); (2) cells that accumulate QD in their volume (28.0 +/- 1.9%), and (3) cells, surrounded by a halo of QD (59.0 +/- 2.2%). The dispersion of these characteristics may suggest the differences in neutrophilic granulocyte plasma membrane permeability.

Contrasting properties of gold nanoparticles for optical coherence tomography: phantom, in vivo studies and Monte Carlo simulation.

The possibility of using silica-gold nanoshells with 150 nm silica core size and 25 nm thick gold shell as contrasting agents for optical coherence tomography (OCT) is analyzed. Experiments on agar biotissue phantoms showed that the penetration of nanoshells into the phantoms increases the intensity of the optical coherence tomography (OCT) signal and the brightness of the corresponding areas of the OCT image. In vivo experiments on rabbit skin demonstrated that the application of nanoshells onto the skin provides significant contrasting of the borders between the areas containing nanoshells and those without. This effect of nanoshells on skin in vivo is manifested by the increase in intensity of the OCT signal in superficial parts of the skin, boundary contrast between superficial and deep dermis and contrast of hair follicles and glands. The presence of nanoshells in the skin was confirmed by electron microscopy. Monte Carlo simulations of OCT images confirmed the possibility of contrasting skin-layer borders and structures by the application of gold nanoshells. The Monte Carlo simulations were performed for two skin models and exhibit effects of nanoparticles similar to those obtained in the experimental part of the study, thus proving that the effects originate exactly from the presence of nanoparticles.